Method for the determination of glycated hemoglobin

ABSTRACT

A method of determining the percentage of glycated hemoglobin in a blood sample is disclosed wherein at least one of the assay steps is performed electrochemically. The method includes determining the total amount of hemoglobin in a sample by electrochemically measuring, in an oxygen electroreduction reaction at a cathode, the amount of oxygen in the sample. Because the amount of oxygen dissolved in the sample is known, the total hemoglobin is determined by subtracting the amount of free oxygen from the total oxygen measured, recognizing the fast equilibrium Hb+O 2 ⇄HbO 2 . This can be followed by determining the amount of glycated hemoglobin in the sample. The cathode reaction is accomplished by contacting the sample with an enzyme, the enzyme being a copper-containing enzyme having four copper ions per active unit. The family of these enzymes includes, for example, laccases and bilirubin oxidases.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority from U.S. provisional patentapplication No. 60/291,361, entitled “Biofuel Cell”, filed May 16, 2001,as well as US provisional application entitled “Miniature BiologicalFuel Cell That is Operational Under Physiological Conditions”, filed May2, 2002, naming inventors Heller, Mano, Kim, Zhang and Mao (underattorney docket M-12761 V1 US), the contents of which applications areincorporated herein by reference in their entireties.

BACKGROUND

[0002] 1. Field of the Invention

[0003] The present invention relates to a process for the determinationof the amount of irreversibly glycated hemoglobin, or HbA1c, present ina sample of blood, relative to the amount of total hemoglobin. Inparticular, the invention incorporates in the method an electrochemical,enzyme-catalyzed reaction or reactions.

[0004] 2. Background Information

[0005] HbA1c is a glycated hemoglobin formed by a binding reactionbetween an amine group of hemoglobin and the glucose aldehyde group, forexample between the amino group of the N-terminal valine of the β-chainof hemoglobin and the glucose aldehyde group. The binding reaction firstforms a Schiff's base and then a stable ketoamine by Amadorirearrangement. The percentage of HbA1c (i.e. the amount of glycatedhemoglobin relative to total hemaglobin in the blood) has come to betaken as a measure of the level of blood glucose control a diabeticpatient has maintained for a period of two or three months prior to themeasurement. As such, percentage HbA1c has become an importantmeasurement by which health care providers can assist diabetic patientsin their care.

[0006] There are many known assays that can be used to determine HbA1cpercentage. In recent years research efforts have focused on creatingassays that are both highly accurate and fast. However, known HbA1cassays typically require a substantial number of time-consuming stepswherein the blood components must be separated and treated.

[0007] In the health care context, a diabetic patient is typicallyguided by a physician to obtain an HbA1c measurement when the physicianrealizes that there is a need for such information during an officevisit. The patient then provides a blood sample to a laboratory andresults are returned to the physician hours or days later. Typically,the lab will use a table top analyzer of the type presently availablecommercially. This time lag between the patient's visit and the resultof the test requires that the physician review the result long after thepatient has left the office. If the physician believes that furtherconsultation with the patient is required in light of the test result,the patient must be contacted again.

[0008] Currently, there is a device sold under the name “A1c NOW” byMetrika, Inc. of Sunnyvale, Calif. This handheld and disposable device(based on technology described in U.S. Pat. No. 5,837,546 entitled“Electronic Assay Device and Method,” incorporated herein by reference)is said to provide an HbA1c test result in eight minutes using arelatively small sample of blood. The A1c NOW device is an example ofthe market demand for a fast method of providing an HbA1c result foreither home or doctor's office use. However, the A1c NOW device is notas accurate as some laboratory assays. Thus, research has continued tofocus on finding a highly accurate HbA1c assay that is also fast enoughand simple enough to permit a diabetic and his or her doctor to take ablood sample during an office visit and have a trustworthy HbA1cmeasurement available for discussion in the same visit.

SUMMARY OF THE INVENTION

[0009] The present invention comprises a method of determining theamount or percentage of glycated hemoglobin in blood or a sample derivedfrom blood, in which at least one of the assay steps is performedelectrochemically. The use of electrochemical methodology can retain orimprove the accuracy of other methods and potentially speed the ultimatedetermination. Devices providing electrochemical measurements can alsobe relatively small.

[0010] In one embodiment, the method includes electrochemicallydetermining the total amount of hemoglobin in a sample byelectrochemically measuring, in an oxygen electroreduction reaction at acathode, the amount of oxygen in the sample, preferably after it wasexposed to air so as to assure that the hemoglobin is oxygenated.Because the amount of oxygen dissolved in aerated physiological bufferat the assay temperature in the absence of hemoglobin, termed here freeoxygen, is known, the total hemoglobin may be determined by subtractingthe amount of free oxygen from the total oxygen measured, recognizingthe fast equilibrium Hb+O₂⇄HbO₂. Electrochemically determining the totalhemoglobin value can be followed by a determination of the amount ofglycated hemoglobin in the sample. In the process of the invention, thecathode reaction is accomplished by contacting the sample with anenzyme. In this embodiment, the enzyme can be a copper-containingenzyme, containing four copper ions per active unit. The family of theseenzymes includes, for example, laccases and bilirubin oxidases.

[0011] The glycated hemoglobin can be determined in different ways. Inone embodiment, the glycated hemoglobin is separated from the sample,for example by capturing it with immobilized antibodies against HbA1c orwith a boronic acid modified surface. Examples of surfaces include thoseof small magnetic, polymer or glass beads. The percentage HbA1c can thenbe determined by either measuring the hemoglobin left in the sample fromwhich the HbA1c has been removed, or by measuring the amount of glycatedhemoglobin in the separated portion of the sample. The amount ofglycated hemoglobin can be measured spectrophotometrically, or by anelectrochemical measurement in the same manner as the total hemoglobin.In another embodiment the hemoglobin is hydrolyzed by an establishedmethod, such as digestion with a proteolytic enzyme. The ketoamines inthe hydrolyzate, such as the fragments comprising the Amadorirearrangement products of the Schiff base formed of amino acids,including valine and glucose, are then determined, preferably by anelectrochemical method. In the electrochemical method, theelectrooxidation of the hydrolyzed Amadori rearrangement product may becatalyzed by an enzyme and a dissolved or immobilized redox mediator.The enzyme can be, for example, a fructosamine oxidase, a fourcopper-ion containing copper enzyme such as a laccase or a bilirubinoxidase, ceruloplasmin, or ascorbate oxidase. The redox mediators canbe, for example, complexes of Os^(2+/3+), or of Ru^(2+/3+).

DETAILED DESCRIPTION

[0012] The invention incorporates one or more electrochemical steps inthe method of determining percentage HbA1c. The method of the inventionis based on the understanding that hemoglobin, being the oxygen carrierof blood, reversibly binds oxygen, forming HbO₂. The equilibriumHb+O₂⇄HbO₂ is rapid. Because O₂ is rapidly released by HbO₂ when O₂ isdepleted from the solution in an electrochemical cell, it is possible todetermine the concentration of HbO₂ in light of the reaction4H⁺+4e⁻+HbO₂→2H₂O+Hb.

[0013] Determining Total Hemoglobin Electrochemically

[0014] In the invention, it may be useful to pre-treat a blood sample bycollecting the relatively large blood cells on a filtration membrane.After rinsing the collected cells with saline to remove adheringproteins, the cell membranes may be ruptured by exposing them todeionized water or a detergent. In this manner, the dissolved hemoglobinwill pass the filtration membrane. The cell membranes will remain on thefilter paper.

[0015] In a preferred form of the invention, total hemoglobin is thendetermined from the sample by electroreducing the oxygen bound to thehemoglobin to water at the cathode in an electrochemical cell. Theoxygen electroreduction catalyst preferably comprises a so-called“copper” enzyme such as bilirubin oxidase, a laccase, or an ascorbateoxidase.

[0016] The catalyst may further comprise a redox mediator to form a“wired enzyme” arrangement. In this system, the electrical connection isbetween a cathode of the electrochemical cell and the oxygen reductioncatalyzing enzyme, especially a copper-containing enzyme, such asbilirubin oxidase (sometimes referenced herein as BOD). Thus, in oneform of the invention, it is preferred to “wire” reaction centers of anenzyme, e.g. bilirubin oxidase, to a cathode. Bilirubin oxidasecatalyzes the four-electron reduction of oxygen to water. A cathodeconstructed with bilirubin oxidase is especially preferred as the redoxenzyme can function under relatively neutral pH conditions. However,other enzymes (e.g. lacasse) may be useful in the method of theinvention so long as they provide catalytic functionality for thereduction of oxygen to water.

[0017] Thus, the concentration of HbO₂ can be measured by the reaction4H⁺+4e⁻+HbO₂→2H₂O+Hb. This measurement may be done coulometrically. Theconcentration of available oxygen in arterial blood tends to be about 8mM. Because the concentration of O₂ in water in equilibrium with air at25° C. is known (the concentration is generally around 0.24 mM), theamount of non-Hb bound O₂ can then be subtracted in calculating theamount of HbO₂.

[0018] A cathode useful in the invention effectuates the four-electronelectroreduction of O₂ to water. The blue, copper-containing oxidases,examples of which include laccases, ascorbate oxidase, ceruloplasmine,and bilirubin oxidase, catalyze the four-electron reduction of O₂ towater. The preferred enzymes are exemplified by bilirubin oxidases,which unlike laccases, retain their more than 80%, and usually retainmore than 90%, of the maximal activity under physiological pH. Thecatalytic reduction of O₂ to water depends on the coordination of thefour Cu^(+/2+) ions of the enzymes. The Cu^(+/2+) ions are classified,by their ligands, into three “types”, types 1, 2, and 3. Type 1Cu^(+/2+) centers show an intense Cys S to Cu(2) charge transfer band ataround 600 nm; the site accepts electrons from an organic substrate,such as a phenol, ascorbate, or bilirubin, and relays the electrons tothe O₂-reduction site. The O₂-reduction site is a trinuclear cluster,consisting of one type 2 Cu^(+/2+) center and a pair of type 3 Cu^(+/2+)centers, their spectrum showing a shoulder at 330 nm.

[0019] There are different forms of bilirubin oxidase available, such asbilirubin oxidase from Myrothecium verrucaria (Mv-BOD) and bilirubinoxidase from Trachyderma tsunodae (TtBOD). Bilirubin oxidases areusually monomeric proteins and have molecular weights approximatelyranging from about 52 kDa to about 65 kDa. Tt-BOD is a monomeric proteinwith a molecular weight of approximately 64 kDa, while that of Mv-BOD isabout 52 kDa. Both Mv-BOD and Tt-BOD are multicopper oxidases, eachcontaining one type 1, one type 2, and two type 3 copper ions. Thesethree types are defined by their optical and magnetic properties. Type 1(blue) copper ions have a characteristic Cys to Cu (2) charge-transferband near 600 nm. The type 1 copper center accepts electrons from theelectron-donating substrate of the enzyme and relays these to the O₂reduction site. The latter is a trinuclear cluster, consisting of a type2 copper ion and a type 3 pair of cupric ions with a characteristic 330nm shoulder in its absorption spectrum.

[0020] In one embodiment of the invention, bilirubin oxidase fromMyrothecium verrucariacould be used in a cathode electrocatalyst layer.In a cathode constructed using Mv-BOD, the electrostatic adduct of thepoly-anionic Mv-BOD and its “wire”, the polycationic redox copolymer ofpolyacrylamide (PAA) and poly (N-vinylimidazole) (PVI) complexed with[Os (4,4′-dichloro-2,2′-bipyridine)₂Cl]^(+/2+), are immobilized on thecathode.

[0021] In another embodiment of the invention, bilirubin oxidase (BOD)from Trachyderma tsunodae can be used in a cathode electrocatalystlayer. In Tt-BOD all of the ligands of the Type 2 and Type 3 Cu^(+/2+)centers are His (histidines), similar to ascorbate oxidase. It isbelieved that the full histidine coordination of the type 2 Cu^(+/2+)center is the underlying cause of the relative insensitivity ofbilirubin oxidases to inhibition by the chloride and hydroxide anions(as are found at physiological concentration). Accordingly, it isexpected that other enzymes having the three types of copper centerswould also be useful as components of cathode electrocatalysts incathodes operating under at near neutral pH.

[0022] The redox potentials of the redox polymers that “wire” thecathode enzyme can be tailored for use in the invention. Redox polymersfor use in the method may includePAA-PVI-[Os(4,4′-dichloro-2,2′-bipyridine)₂Cl]^(+/2+) which can beprepared as follows: 4,4′-Dinitro-2,2′-bipyridine N,N′-dioxide wasprepared as described in Anderson, S.; Constable, E. C.; Seddon, K. R.;Turp, E. T.; Baggott, J. E.; Pilling, J. J. Chem. Soc., Dalton Trans.1985, 2247-2250, and Kenausis, G.; Taylor, C.; Rajagopalan, R.; Heller,A. J. Chem. Soc., Faraday Trans. 1996, 92, 4131-4135.4,4′-dichloro-2,2′-bipyridine (dcl-bpy) was synthesized from4,4′-dinitro-2,2′-bipyridine N,N′-dioxide by modifying the procedure ofMaerker et al. (see Anderson, S., supra and Maerker, G.; Case, F. H. J.Am. Chem. Soc. 1958, 80, 2475-2477.). Os(dcl-bpy)₂Cl₂ was prepared asfollows: (NH₄)₂OsCl₆ and “dcl-bpy were dissolved in ethylene glycol in a1:2 molar ratio and refluxed under argon for 1 hour (yield 85%). TheOs(dcl-bpy)₂Cl₂ was then complexed with the 1:7polyacrylamide-poly(N-vinylimidazole) (PAA-PVI) copolymer and purifiedas described in Zakeeruddin, S. M.; D. M. Fraser, D. M.; Nazeeruddin, M.-K.; Gratzel, M. J. Electroanal. Chem. 1992, 337, 253-256 to form thePAA-PVI-[Os(4,4′-dichloro-2,2′-bipyridine)₂Cl]^(+/2+) redox polymer.Those skilled in the art are aware of numerous variations that can beprepared and used as redox polymers according to the invention.

[0023] Determination of the HbA1c Percentage

[0024] Once the total hemoglobin has been measured, the HbA1c/Hb ratiocan be determined by separating the HbA1c fraction from the sample. TheHbA1c, which can be converted to HbA1cO2, can then be measuredindirectly and electrochemically using the same method as for the totalhemoglobin. Alternatively, these fructosyl amines may be subject todirect enzyme catylized electro-oxidation, for example using fructosylamine oxidases having FAD/FADH reaction centers, or by one of the copperenzymes.

[0025] The following are examples of suitable methods which incorporatethe separation and HbA1c assay steps.

EXAMPLE 1

[0026] Affinity gel columns can be used to separate bound, glycosylatedhemoglobin from the nonglycosylated fraction. The gel containsimmobilized m-aminophenylboronic acid on cross-linked, beaded agarose.The boronic acid first reacts with the cis-diol groups of glucose boundto hemoglobin to form a reversible 5-membered ring complex, thusselectively holding the glycosylated hemoglobin on the column. Next, thenonglycosylated hemoglobin is eluted. The ring complex is thendissociated by sorbitol, which permits elution of the glycosylatedhemoglobin. Using affinity chromatography, absorbances of the bound andnonbound fractions, measured at 415 nm, are used to calculate thepercent of glycosylated hemoglobin.

EXAMPLE 2

[0027] Magnetic beads that are <1 μm (available from BangsLaboratories), on which antibodies against HbA1c would be immobilized,can be mixed with a citrate-solution diluted blood sample. Twomeasurements are performed, one on the entire sample, and a second onthe re-oxygenated Hb1Ac bound to the magnetic beads, after their removalto a chamber of an electrochemical cell. Alternatively, the secondmeasurement can be on the residual Hb, after the magnetic separation ofthe bead-bound HbA1c.

EXAMPLE 3

[0028] Two samples of the lysed red blood cells in citrate buffer can becoulometrically assayed in two chambers. In Chamber 1, the total HbO₂would be measured. Chamber 2 contains the immobilized HbA1c-specificantibody. Either of the two would capture HbA1c without capturing Hb.After rinsing or passage of citrate buffer through Chamber 2 (e.g. byrepeated filling through capillary action and touching the edge of thechamber to filter paper), the chamber would contain only HbA1cO₂. TheHbA1cO₂ would be assayed electrochemically (preferably coulometrically)by its electroreduction, 4H⁺+4e⁻+HbA1cO₂→2H₂O+HbA1c. The HbA1c/Hb ratiocan then be calculated from the two coulometric measurements.

EXAMPLE 4

[0029] As in example 3 above, except that the two coulometricmeasurements would be performed in a single chamber. The chamber, whichwould contain the immobilized HbA1c capture agent, would be filled witha citrate solution of the lysed red blood cells. First, the total HbO₂would be electrochemically (preferably coulometrically) measured. Next,the unbound Hb, but not the bound HbA1c, would be rinsed out, the HbA1cwould be re-equilibrated with air, and its amount would becoulometrically measured.

[0030] Thus, the assay of the invention, in one form, can comprise amethod of determining the ratio of HbA1c to total Hb in blood, themethod comprising obtaining a blood sample; electrochemicallydetermining the total amount of hemoglobin in the sample, or in atreated portion of the sample; electrochemically determining the amountof HbA1c in the sample; and calculating the ratio of HbA1c to totalhemoglobin. In a preferred form the method of electrochemicallydetermining the total amount of hemoglobin in the sample is accomplishedby placing the sample in an electrochemical cell in which, at thecathode, a cathode enzyme is bound, for example using a redox polymer.In this method, it is preferred that the enzyme be a laccase or abilirubin oxidase which will electroreduce oxygen bound to thehemoglobin to water. The hemoglobin content is determined from theoxygen content.

[0031] In another form of the invention the electrochemicaldetermination of HbA1c fraction can be accomplished by one of twomethods. In a first method, the A1c containing fraction of thehemoglobin is separated by physical means, such as by use of an HbA1cspecific antibody. Under appropriate conditions the HbA1c then presentin the form of HbA1cO₂ can then be electrochemically determined byelectroreduction of the oxygen (again with an enzyme selected toaccomplish the four electron reduction of oxygen). In a second method,the glycated protein (a fructosyl amine) can be directly oxidized oncross-linked poly(N-vinyl imidazole) based redox polymer films (withoutan enzyme) of sufficiently positive oxidizing potential. Alternatively,enzymatic electrooxidation of the fructosyl amines can be used for thispart of the determination.

[0032] Finally, the invention comprises an electrochemical method forthe determination of HbA1c (or HbA1c/Hb ratio) comprising determiningfrom a starting sample, in an electrochemical cell, the total amount ofhemoglobin (e.g. by measuring bound oxygen), separating the HbA1ccomponent from the sample using an HbA1c capturing agent, and measuringhemoglobin content in the captured or non-captured portion of thesample.

[0033] Devices for accomplishing the method of the invention arepreferably small. By incorporating electrochemical steps, it may bepossible to prepare biosensor strips which include a cathode at whichthe chemistry discussed herein is placed, as well at which the necessaryanode is constructed. Such strips can be prepared using techniquespresently used for making commercially available biosensor strips thatare used for glucose determinations, such as the FreeStyle blood glucosesystem sold by TheraSense, Inc. Samples could then be applied to thesestrips and the strips placed in the measuring instrument (meter) to be“read.” By constructing a portion of the equipment in the form ofelectrochemical biosensor strips, the electrochemical method of theinvention provides a significant potential advantage of creating asmaller analysis device while providing accurate results.

What is claimed is:
 1. A method for determining the percentage ofglycated hemoglobin in a fluid sample comprising: electrochemicallydetermining the total amount of hemoglobin in a blood sample;determining the amount of glycated hemoglobin in the sample; andcalculating the ratio or percentage of glycated hemoglobin to totalhemoglobin.
 2. The method of claim 1, wherein electrochemicallydetermining the total amount of hemoglobin comprises: contacting thesample in an electrochemical cell with an enzyme capable of determiningthe oxygen content of the sample; and calculating the amount ofhemoglobin from the oxygen content.
 3. The method of claim 2, whereinthe enzyme is a copper-containing enzyme.
 4. The method of claim 2,wherein the enzyme is selected from the group consisting of laccase,bilirubin oxidase, and ascorbate oxidase.
 5. The method of claim 1,wherein determining the amount of glycated hemoglobin is performedspectrophotometrically.
 6. The method of claim 1, wherein determiningthe amount of glycated hemoglobin is performed electrochemically.
 7. Themethod of claim 1, wherein determining the amount of glycated hemoglobinis performed by direct enzymatic action.
 8. The method of claim 1,wherein determining the amount of glycated hemoglobin is performed bydirect enzymatic reaction.
 9. A method for determining the percentage ofglycated hemoglobin to total hemoglobin in blood comprising:electrochemically determining the total amount of hemoglobin in a bloodsample; separating the glycated hemoglobin from the sample to form asecond sample; determining the amount of glycated hemoglobin in thesecond sample; and calculating the percentage of glycated hemoglobin tototal hemoglobin in the sample.
 10. The method of claim 9, whereindetermining the amount of glycated hemoglobin in the second sample isdone electrochemically.
 11. The method of claim 9, wherein determiningthe amount of glycated hemoglobin in the second sample is donespectrophotometrically.
 12. A method for determining the percentage ofglycated hemoglobin to total hemoglobin in blood comprising:electrochemically determining the total amount of hemoglobin in a bloodsample by exposing the sample to an enzyme associated with a cathode,determining the amount of oxygen from an enzymatic redox reaction, andcalculating the amount of hemoglobin from the amount of oxygen;determining the amount of glycated hemoglobin in the sample; andcalculating the percentage of glycated hemoglobin to total hemoglobin inthe sample.